The biosynthesis of covalent crosslinkages in elastin and collagen is fundamental to the proper development and function of connective tissues. Lysyl oxidase is the enzyme which can initiate crosslink formation by catalyzing the deamination of lysyl residues of these proteins to yield peptidyl gamma-aminoadipic-delta-semialdehyde. Therefore, this enzyme potentially plays a controlling role in the development, aging and repair of blood vessels and other connective tissues whose elasticity and tensile strength reflect their content of crosslinked elastin and collagen fibers. Although considerable investigate effort has yielded much information about the chemistry and the biosynthesis of crosslinkages, much remains to be accomplished to understand fully the role of lysyl oxidase in this process in both healthy and diseased connective tissues. One of the major efforts of this proposal will be to develop a quantitative, chemically defined and sensitive assay system which will greatly facilitate important mechanistic studies on the enzyme as well as assessment of growth-and disease-related changes in enzyme activities. Toward this end, we will chemically synthesize lysine-containing polypeptides of defined but varied sequence and length to be tested as substrates. Naturally-occurring soluble and insoluble forms of elastin and collagen will also be isolated and used as substrates. The use of such substrates and location of product aldehydes generated by enzyme will be essential in defining the substrate specificity of lysyl oxidase, and in "mapping" the active site of this enzyme. We will also test the possibility that there may be molecularly distinct enzyme forms, as our present studies with aorta and ligament data suggest, and that there may be elastin- and collagen- specific species of lysyl oxidase. We will explore the substrate requirements and physical-chemical properties of purified enzymes from aorta, ligament and cartilage in efforts to distinguish and mechanistically probe the isolated enzymes.